G proteins transduce extracellular signals, detected by cell surface receptors for hormones or sensory stimuli, into cellular responses mediated by altered function of effector molecules (enzymes or ion channels). Gs, the best characterized member of the G protein family, mediates hormonal stimulation of adenylyl cyclase and opening of Ca2+ channels. The alpha chain of Gs, called alpha s, binds and hydrolyzes GTP and carries structural determinants for specific interactions with receptors and adenylyl cyclase. This proposal asks two related questions: 1. How do the molecular structures of G protein alpha chains in conformation, and interactions with receptors, effectors, and beta gamma? 2. How does the cell regulate expression, abundance, assembly, subcellular location, and signaling functions of G proteins? To answer the first question, we will assess the functions of mutant alpha s proteins - produced by site-directed mutagenesis, random linker insertion-deletions, random mutagenesis of localized regions, and construction of chimeras between different alpha chains - expressed in alpha s-deficient S49 cyc- cells. To answer the second question, we will: Isolate monoclonal antibodies (mAbs) directed against native alpha s; use these antibodies to assess rates of synthesis and turnover of metabolically labeled alpha s; assess binding of alpha s to beta gamma, receptors, adenylyl cyclase, and protein components of the submembranous cytoskeleton, by immunoprecipitating complexes of alpha s with other proteins, and with the help of bifunctional cross-linking agents. Experiments aimed at answering each class of question will provide reagents and information useful in tackling the other class of question: alpha s mutations will help to determine how function of an alpha s molecule (e.g., its ability to hydrolyze GTP or to take on a GTP- dependent."active" conformation) alters its membrane targetting and associations with other proteins, while anti-alpha s mAbs will be essential for identification and biochemical analysis of wild type and mutant alpha s chains.